Solubilization and characterization of vitamin K epoxide reductase from normal and warfarin-resistant rat liver microsomes

Two procedures have been developed for the solubilization of vitamin K epoxide reductase from rat liver microsomal membranes using the detergent Deriphat 160 at pH 10.8. The methods are applicable to both normal and Warfarin-resistant-strain rat liver microsomes and yield material suitable for further purification. The preparations retain dithiothreitol-dependent vitamin K quinone reductase activity as well as vitamin K epoxide reductase and are free of vitamin K-dependent carboxylase and epoxidase activities. Optimal epoxide reductase activity is obtained at 0.1 M KCl and pH 9 in the presence of sodium cholate. Artifactual formation of vitamin K metabolites was eliminated through the use of mercuric chloride to remove excess dithiothreitol prior to extraction and metabolite assay. Using the solubilized enzyme, valid initial velocities were measured, and reproducible kinetic data was obtained. The substrate initial velocity patterns were determined and are consistent with a ping-pong kinetic mechanism. The kinetic parameters obtained are a function of the cholate concentration, but do not vary drastically from those obtained using intact microsomal membranes. At 0.8% cholate, the enzymes solubilized from normal Warfarin-sensitive- and Warfarin-resistant-strain rat livers exhibit respective values of Vmax = 3 and 0.75 nmol/min/g liver; Km for vitamin K epoxide = 9 and 4 microM; and Km for dithiothreitol of 0.6 and 0.16 mM.     

Normal and warfarin-resistant rat hepatocyte metabolism of vitamin K 2,3-epoxide: evidence for multiple pathways of hydroxyvitamin K formation

Vitamin K and 3- (and/or 2)-hydroxy-2,3-dihydro-2-methyl-3-phytyl-1,4-naphthoquinone (hydroxyvitamin K) have been identified as metabolites of vitamin K 2,3-epoxide incubated with hepatocytes isolated from normal and warfarin-resistant rats. Dithiothreitol added to the extracellular medium differentially enhanced the formation of both metabolites: hydroxyvitamin K formation, almost undetectable in the absence of dithiothreitol, was particularly affected. Addition of the vitamin K 2,3-epoxide reductase inhibitors warfarin (5 to 100 microM) and brodifacoum (1 to 5 microM) to normal rat hepatocyte cultures produced a slight increase in hydroxyvitamin K formation and a marked inhibition of vitamin K formation. Brodifacoum was a weak inhibitor of hydroxyvitamin K formation at higher concentrations. Hepatocytes from warfarin-resistant rats catalyzed hydroxyvitamin K formation 1.5 to 2 times faster and vitamin K formation 1.5 to 2 times slower than did normal rat hepatocytes. The addition of warfarin to these cultures had no effect on epoxide metabolism to hydroxyvitamin K and only partially diminished metabolism to vitamin K. In contrast, brodifacoum (1 microM) addition produced 50% inhibition of hydroxyvitamin K formation and almost complete inhibition of vitamin K formation. These data suggest that in resistant, but not in normal rat hepatocytes, the vitamin K 2,3-epoxide reductase makes a significant contribution to hydroxyvitamin K formation. A second sulfhydryl-dependent pathway, present in both strains, is also involved in the formation of this metabolite. They also suggest that in resistant rats, warfarin inhibition of the vitamin K 2,3-epoxide reductase, and presumably the sulfhydryl-dependent vitamin K reductase, is incomplete and independent of concentration.     

Formation of hydroxyvitamin K by vitamin K epoxide reductase of warfarin-resistant rats

A new metabolite of vitamin K, 2(3)-hydroxy-2,3-dihydro-2-methyl,3-phytyl-1,4-naphthoquinone (hydroxyvitamin K), has been identified as a product of vitamin K epoxide metabolism in hepatic microsomes from warfarin-resistant rats, but not in those derived from normal rats. The structure was determined by comparison of the high performance liquid chromatography retention times, UV, IR, CD, and mass spectra of the unknown with chemically synthesized standards. Alterations in the formation of hydroxyvitamin K occur in parallel with alterations in total vitamin K epoxide conversion with respect to reaction time, extent of reaction, detergent stimulation, and inhibition by warfarin. Thus, hydroxyvitamin K appears to be a product of the warfarin-resistant vitamin K epoxide reductase. It is neither a substrate nor an inhibitor of epoxide reduction. Hydroxyvitamin K is formed from both enantiomers of racemic vitamin K epoxide with little stereoselectivity for the configuration of either the oxirane ring or the phytyl side chain. The reaction is stereospecific; however, the biologically formed (+)-vitamin K epoxide yields exclusively (+)-3-hydroxyvitamin K. Observation of this product is discussed as a key to understanding the normal reaction mechanism of the enzyme.     

Vitamin K dependent carboxylase: subcellular location of the carboxylase and enzymes involved in vitamin K metabolism in rat liver

Vitamin K dependent carboxylation of an exogenous peptide substrate and endogenous protein substrates, vitamin K epoxidation, and reduction of vitamin K epoxide were measured in subcellular fractions from rat liver. The rough microsomal fraction was highly enriched in all four activities; lower levels were found in smooth microsomes. Mitochondria, nuclei, and cytosol had negligible activities. The addition of 0.2% Triton X-100 to intact microsomes resulted in a 10-20-fold stimulation in carboxylation of a peptide substrate. This marked latency suggests that the active site of the carboxylase may be accessible only from the lumen of the microsomal membrane. A lumen-facing orientation of the carboxylase was also supported by its inaccessibility to trypsin in intact microsomes contrasted with marked inhibition by trypsin in detergent-permeabilized microsomes. Vitamin K epoxidase and epoxide reductase activities were also inhibited by trypsin much more effectively in permeabilized than in intact microsomes, although some degree of exposure at the cytosolic surface was also indicated. These data suggest that carboxylation is an early event in prothrombin synthesis occurring primarily on the lumen side of the rough endoplasmic reticulum membrane. The location of the vitamin K epoxidation-reduction cycle enzymes is consistent with their possible role in the carboxylation reaction.     

Caffeine: its action on purine metabolizing enzymes.

The many pharmacological and biochemical effects of caffeine may be explained in part by its inhibitory action in vivo and in vitro upon enzymes which metabolize purines. We have demonstrated that in $\text{Crithidia}\text{fasciculata}$ this methylxanthine (as well as theophylline) is a rather weak competitive inhibitor of adenine aminohydrolase, ribonucleoside hydrolase, hypoxanthine and guanine phosphoribosyltransferases. Caffeine does not interfere with purine base transport in Crithidia, however in leucocytes purine uptake is reduced. While the methylxanthines are weak purine enzyme inhibitors, the large number of enzymes affected accounts for their physiological importance in these cells.     

Induction of drug metabolizing enzymes by vitamin E

Vitamin E is an essential micronutrient involved in various processes relevant to human health and disease. Although it has long been considered just as an antioxidant, it has now become clear that vitamin E has functions far exceeding that as an antioxidant. These include regulation of cellular signaling processes and gene expression. Expression control of enzymes involved in drug metabolism was recognized during the investigation of vitamin E degradation. Vitamin E is metabolized by side chain degradation initiated by an omega-hydroxylation, catalyzed by a cytochrome P450 enzyme (CYP). This mechanism is identical for all forms of vitamin E. The degree to which they are degraded, however, varies dramatically, and may, in part, explain their different biological activities. CYPs degrade various endogenous and exogenous compounds and many of them are induced by their substrates. Also, gamma-tocotrienol, identified as substrate of CYPs, increased endogenous CYP3A4 in human HepG2 cells. In two studies with mice undertaken independently, alpha-tocopherol induced Cyp3a11, the murine homolog to human CYP3A4, whereas neither gamma-tocopherol nor gamma-tocotrienol, due to rapid degradation, showed any effect. CYPs are induced via the activation of the pregnane-X-receptor (PXR), a member of the family of nuclear receptors. They are activated by a large number of lipophilic xenobiotics. Also, vitamin E induced a reporter gene driven by PXR. The induction was highest with alpha- and gamma-tocotrienol and low but significant with alpha-tocopherol. This roughly correlates with the in vitro binding of vitamin E to PXR. These findings reveal that, in principle, vitamin E is able to directly influence gene activity. They also raise the question of whether vitamin E may interfere with drug metabolism in humans. Related research is urgently deeded.     

Cadmium sensitivity differences between liver microsomal drug metabolizing enzyme systems of guinea-pig and rat

1. Male guinea-pigs (400-500 g) and rats (225-275 g) were given a single dose of cadmium chloride (CdCl2) (2 mg Cd2+/kg i.p.) and 72 hr later the liver microsomal drug metabolizing enzyme activities and Cd levels of tissues and microsomes were determined. 2. No significant differences were noted between Cd treated and control animal tissue weights of microsomal protein contents in either guinea-pigs or rats. 3. Cd treatment exhibited significant inhibition of the activities of aniline 4-hydroxylase and ethylmorphine N-demethylase and on the levels of cytochrome P-450 and cytochrome b5 of liver of both species but the degree of inhibition were not the same in the species; they were 23, 34, 16 and 10% in guinea-pigs and 58, 57, 25 and 13% in rats, respectively. 4. No activity changes were observed in liver NADPH-cytochrome c reductase of the species by Cd treatment. 5. The duration of hexobarbital sleeping time was significantly prolonged in both species. However, the prolongation was 1.6 fold in guinea-pigs but 3.4 fold in rats. 6. No significant differences were found between either tissue or microsomal Cd levels of guinea-pigs and rats.     

Ontogeny of steroid metabolizing enzymes in rat oligodendrocytes

Rat oligodendroglial cells were isolated from newborn and developing brains and used immediately after, for quantification of steroid metabolizing activities. Oligodendrocytes (Ol) and their progenitor cells were incubated with [(14)C] testosterone, [(14)C] progesterone, [(14)C] pregnenolone or [(14)C] dehydroepiandrosterone (DHEA). Oligodendrocytes and their progenitor cells expressed different steroid metabolizing enzymes. The main activities were 5 alpha reduction of testosterone and progesterone and 3 beta hydroxy steroid dehydrogenase-isomerase which transformed pregnenolone into progesterone and DHEA into Delta 4 androstenedione. 5 alpha reductase activity increased in male and female rats in parallel with testosterone or progesterone. Contrary to this, 3 beta hydroxysteroid dehydrogenase-isomerase activity was found to be high in the young rat and to decrease when testosterone and progesterone plasma concentration increased.     

Effect of chlorinated naphthalenes and terphenyls on the activities of drug metabolizing enzymes in rat liver

γ-Aminobutyric acid-α-ketoglutarate transaminase from pig brain is irreversibly inactivated by 4-amino-5-halopentanoic acids. Protection from inactivation by the natural substrates, the pH dependence of inactivation and the incorporation of 1.7 moles of radioactive inhibitor per mole of enzyme from (S)-[U-¹⁴C]-4-amino-5-chloropentanoic acid suggest a covalent adduct at the active site of the enzyme. A mechanism-based inactivation is proposed.     

Effect of natural and structurally modified bile acids on cholesterol metabolizing enzymes in rat liver microsomes

The effect of chenodeoxycholic (CDCA), ursodeoxycholic (UDCA), tauroursodeoxycholic (TUDCA), cholic (CA), ursocholic (UCA) acids, analogues of CDCA and UDCA with a cyclopropyl ring at C22, C23 (cypro-CDCA and cypro-UDCA) and 23-methylursodeoxycholic acid (MUDCA) on cholesterol 7 alpha-hydroxylase was studied in rat liver microsomes. Cypro-analogues consisted of a mixture of four diasteroisomers, while MUDCA was the racemic mixture of two enantiomers. Each steroid was added to liver microsomes at concentrations ranging from 10 to 200 microM. With the exception of UCA and CA, all the bile acids inhibited cholesterol 7 alpha-hydroxylase activity. The inhibition shown by cypro-CDCA and cypro-UDCA was stronger than that observed with the corresponding natural compounds. 22S,23S cypro-UDCA exhibited an inhibitory effect which was more pronounced than that of the diasteroisomer mixture. The isomer 22R,23S was less effective and decreased cholesterol 7 alpha-hydroxylase activity in a manner comparable to that of UDCA. The effect of CDCA, UDCA and the cyclopropyl analogues was also tested with respect to HMG-CoA reductase and acylCoA cholesterol acyltransferase (ACAT) activities. ACAT was stimulated by the isomer 22S,23S cypro-UDCA but not affected by the other bile acids. No effect was observed as regards HMG-CoA reductase.     

Effects of maternal diabetes on the development of carbohydrate metabolizing enzymes in fetal rat liver

Effects of streptozotocin-induced maternal diabetes on fetal hepatic carbohydrate-metabolizing enzyme development and hormonal status has been explored in the rat. Hepatic glycogen synthase a activity of the normal fetus rose to a maximum at 20 days of gestation, then fell prior to parturition. In fetuses of diabetic mothers, this prepartum decline was curtailed, resulting in enhanced synthase a activity and increased glycogen content in fetal livers at term. Elevation in hepatic synthase a in fetuses of diabetic mothers was due, not to altered interconversion between existing synthase a and b, but to equivalent increases in both forms of the enzyme. Both hepatic and free plasma corticosterone levels were elevated in fetuses of diabetic mothers and may be responsible for the enhanced development of total glycogen synthase observed in these fetuses. In normal fetuses hepatic phosphofructokinase and pyruvate kinase activities also rose to maxima at 20 days, then declined prior to term. In fetuses of diabetic mothers pyruvate kinase activity attained higher than normal maximal levels and phosphofructokinase activity fell more gradually, thus resulting in elevations in both enzyme activities at term. Augmentations in these glycolytic enzymes are compatible with hyperinsulinemia observed in fetuses of diabetic mothers. The following conclusions may be drawn from these findings. During late fetal life developmental patterns of rate-limiting hepatic glycogen-synthesizing and glycolytic enzymes are adapted to glucose utilization. In the normal fetus these patterns reverse at term, thereby promoting glucose mobilization, which prepares the fetus for abrupt deprivation of maternal glucose at birth. Maternal diabetes results in retardation of these reversal processes, presumably due to elevations in fetal glucocorticoid and insulin levels. Glycogenolytic and glucogenic capacities are thereby impaired in these fetuses.     

Activity of enzymes participating in xenobiotic metabolism and the condition of microsomal membranes of rat liver in vitamin K deficiency

Alimentary deficiency or vitamin K (vitamin K-poor diet) as well as the vitamin deficiency resulting from sinkumar administration are accompanied by a decreased activity of microsomal demethylases, hydroxylase, NADH- and nNADPH-reductases of dichlorophenolindophenol and neotrazolium. The activity of cytosolic enzymes (only glutathione-S-transferases, aryl- and allyl esterases) is diminished in a lesser degree. Vitamin K deficiency does not significantly interfere with the effect of the xenobiotic metabolism enzyme inducer (phenobarbital) or the cytochrome P-450 inhibitor (cobalt chloride). The changes in the enzyme activity result in a decrease of acetanilide biotransformation. A possible reason for the observed changes in the activity of microsomal enzymes is the weakening of hydrophobic and polar interactions in microsomal membranes. This hypothesis was confirmed by experiments with the use of membrane perturbants as well as by solubilization of membrane-bound enzymes.     

Fructose metabolizing enzymes from mouse liver: influence of age and caloric restriction

The influence of caloric restriction (CR) on the activities of liver fructose metabolizing enzymes and metabolite levels were studied in young (3 months) and old (30 months) mice. Fructokinase activity was increased (P<0.05) in both young and old CR mice when compared to controls while triokinase activity was increased (P<0.05) only in old CR versus control mice. Aldolase was not altered by CR in either old or young mice. No age-related differences in activities were observed in controls although a trend towards an increase was observed for triokinase, while significant age-related increases were observed for fructokinase and triokinase, but not aldolase, in CR mice. Both young and old mice on CR showed significant decreases in fructose and fructose-1-phosphate, however, no age-related changes in metabolite levels were observed for either control or CR mice. A fructose-1-phosphate kinase activity was also measured and found to be unchanged in both young and old mice on CR, but the activity was significantly lower in the old mice compared with young. We show here that the enzymes involved in fructose metabolism are influenced by CR and that this could contribute to alterations in gluconeogenesis and glycolysis observed with CR.